Jimenez J, Alphey L, Nurse P, Glover D M
Department of Biochemistry, University of Oxford, UK.
EMBO J. 1990 Nov;9(11):3565-71. doi: 10.1002/j.1460-2075.1990.tb07567.x.
We have exploited the universality of the molecular mechanisms that control entry into mitosis to clone the Drosophila melanogaster homologues of fission yeast Schizosaccharomyces pombe cell division control (cdc) genes by the complementation of temperature sensitive mutations. The Drosophila genes were expressed in S.pombe as cDNAs from the SP6 promoter. Successful recovery of complementing plasmids required that we first 'adapt' pooled plasmids from a Drosophila embryonic cDNA library for propagation in fission yeast by introducing an ars1-LEU2 DNA fragment into the vector. This library was introduced into S.pombe cdc2 and cdc25 mutants, and plasmids isolated carrying cDNAs that complement these mutations. The gene that encodes the Drosophila cdc2 homologue maps to a single locus in the Drosophila genome at 31E on chromosome 2. It is expressed maternally to provide mRNA in syncytial embryos, and appears to be zygotically expressed in mitotically active regions of the cellularized embryo. We have isolated two different cDNAs that complement cdc25-22. One corresponds to a transcript of string, previously described as the Drosophila homologue of cdc25, and the other to a gene that has not been previously characterized.
我们利用控制进入有丝分裂的分子机制的普遍性,通过温度敏感突变的互补作用,克隆了粟酒裂殖酵母裂殖酵母细胞分裂控制(cdc)基因的果蝇黑腹同源物。果蝇基因在粟酒裂殖酵母中作为来自SP6启动子的cDNA进行表达。成功回收互补质粒要求我们首先通过将ars1-LEU2 DNA片段引入载体,“改造”来自果蝇胚胎cDNA文库的混合质粒,以便在裂殖酵母中繁殖。将该文库引入粟酒裂殖酵母cdc2和cdc25突变体中,并分离携带互补这些突变的cDNA的质粒。编码果蝇cdc2同源物的基因定位于果蝇基因组中位于2号染色体31E处的单个位点。它在母体中表达以在合胞体胚胎中提供mRNA,并且似乎在细胞化胚胎的有丝分裂活跃区域中合子表达。我们分离出了两种不同的cDNA,它们与cdc25-22互补。一种对应于string的转录本,先前被描述为cdc25的果蝇同源物,另一种对应于一个先前未被表征的基因。